Bacterial Protein Expression, Purification, and Characterization
Objective/Introduction: Biotechnology has greatly progressed in the last few decades especially in the field of molecular genetics. Technology has been used for protein expression to obtain amplified quantities of proteins that were once previously difficult to acquire. E. coli is often the bacteria of preference as it is harmless to adults. Bacteria are used as expression hosts by transforming the protein of interest in a plasmid vector for the bacteria to incorporate in their own DNA. Bacteria are used for gene expression to amplify the protein of interest[1]. There are several purification strategies to isolate the protein. One such strategy is chromatography in which the solution containing the protein flows through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated to elute the protein from the column. Other protein purification methods include ultracentrifugation and extraction. Expanding technology has clearly led to numerous methods to allow for protein production and purification[2].
In these labs, the purpose to overexpress a recombinant protein (gbr22 purple protein) in bacteria, purify the protein, and use a gel electrophoresis to analyze the protein samples. The importance of these labs is that it builds a foundation to amplify and purify proteins that could be used as potential drug targets in the Virtual Drug Screening research stream.
Materials and Methods: Several safety precautions were taken during these labs. Gloves, goggles, and lab coats were used throughout because toxic chemicals were used. Also, all biohazardous waste was treated and disposed of properly.
In the first part of this lab, the first step was to transform E. coli bacterial cells with gbr22 protein. 3 plates were used including, one experimental plate with DNA, one Control plate that has no DNA, and one plate for ‘fun’ with bacteria from coughing on the plate. The SOC mixture that either contained the bacteria or did not was accordingly added to the appropriate plate. The starter culture was then grown using LB that was supplemented with 100 mg/mL ampicillin. The starter cultures were transferred to Erlenmeyer flasks and set in the incubator to grow over night. After the cultures were purple, the cells were harvested. Next the cells were resuspended in phosphate buffered saline by adding lysozyme to a final concentration of 1 mg/ml.
In the second portion of the lab, the protein was purified. First the E. coli were lysed with the lysozyme added in the first part of the lab. The lysate was then clarified and labeled as sample 2. The lysate was then syringe filtered to discard large particulate matter. The protein bounded to the resin using Ni-NTA resin/buffer mix. The resin and buffer were then transferred to column chromatography and the resulting sample was labeled as sample 3. The Ni-NTA was then washed through the column with the wash buffer and the resulting sample was labeled as sample 4. The gbr22 protein was released from the Ni-NTA by running the column with the elution buffer. The resulting sample was labeled as Elution 1 and the process was run again to obtain Elution 2. The Nanodrop spectrophotometer was used to estimate the concentration of the final purified protein.
In the last portion of the lab, the protein was characterized. The SDS-page gel samples were prepared and assembled in the electrophoresis module. The wells were loaded using a needle and syringe to clear out the wells and a micropipette to add the samples. Next, the gel was stained by adding imperial protein stain. After a short waiting period, the gels were destained and placed in the orbital shaker overnight. The gel was placed on Whatman filter and Saran Wrap. Lastly, the gel was dried in a vacuum in the Biotech lab.
Results:
Figure 1A: AMP Plate with transformed bacteria. The bacteria correctly transofrmed the DNA because they are ampicillin resitant. However, the protein for changing the bacteria to purple is currently not being expressed.
Figure 1B: AMP control plate with no bacteria. The control plate has no bacteria because they were not given the DNA, which means they did not have the ampicillin resistant gene. As a result they could not grow on the plate.
Figure 1C: Fun plate without AMP. The plate was coughed on but no bacteria appeared to have grown.
Figure 2A: Culture #1 of purple bacteria after shaking in the incubator overnight
Figure 2B: Culture #2 of purple bacteria after shaking in the incubator overnight
Figure 3A: Cell pellet of culture #1 that weighed 0.36 g
Figure 3B: Cell pellet of culture #1 that weighed 0.24 g
Figure 4: Elution 1 and Elution 2 in protein purification lab. Elution 1 is slightly purple while Elution 2 is clear.
Figure 5A: Absorbance spectrum of protein at 280 nm for trial #1. The absorbance at 280 nm was found to be 0.225.
Figure 5B: Absorbance spectrum of protein at 280 nm for trial #2. The absorbance at 280 nm was found to be 0.220.
Figure 6A: Absorbance spectrum of protein at 574 nm (maximum wavelength) for trial #1. The absorbance at 574 nm was found to be 0.16 with a pathlength of 1 cm.
Figure 6B: Absorbance spectrum of protein at 574 nm (maximum wavelength) for trial #2. The absorbance at 574 nm was found to be 0.25 with a pathlength of 1 cm.
Figure 7A: Preliminary image of gel before drying process. Well 2 and 9 show the protein ladder. Well 7 includes elution #1 which shows a distinct band that is more than likely the protein of study. Well 8 includes elution #2 and is very faint.
Figure 7B: Gel after drying process. The gel broke because the vacuum was opened during the run without being turned off. This resulted in a pressure changing causing the gel to break.
Sample 1 (Well 3 and 10): Cell Lysate Sample 2 (Well 4): After Centrifugation Sample 3 (Well 5): Flow Through Sample 4 (Well 6): Wash Sample 5 (Well 7): Elution 1 Sample 6 (Well 8): Elution2 Protein Ladder (Well 2 and 9)
Figure 8: Fermentas Page Ruler Prestained Protein Ladder
Figure 1 shows the three plates from part 1 of the lab including the control plate, experimental plate, and fun plate. No bacteria grew on the control plate as intend. Bacteria colonies are present in the experimental plate but they are not seen as purple because they were not expressing the purple protein at the time. The fun plate failed to grow any bacteria on it. Figure 2 shows the two cultures of purple bacteria that grew in the incubator overnight. Figure 3 shows the cell pellet after centrifugation. Figure 3A shows a protein pellet that weighs 0.36 g and Figure 3B shows a protein pellet that weighs 0.24 g. Figure 4 shows Elution 1 and 2 acquired after protein purification. Figure 5 and 6 shows the absorbance spectrum at 280 and 574 nm for the protein. At 280 nm the yield for the average of the trials was 0.662 mg. The yield for the maximal wavelength (574 nm) was 0.201 mg.
Beers Law: A = εlc
Yield for 280 nm Wavelength A = Average absorbance at 280 nm = (0.22 + 0.225) / 2 = 0.2225 ε = Extinction Coefficient = 39100 M^-1 cm^-1 l = Path Length = 1 cm Molecular Weight = 25794.2 g/mol
Volume of protein in Elution #1 = 4.5 mL Yield = (4.5 mL) (0.0447 mg/mL) Yield = 0.201 mg
Figure 7A show the gel before it is dried. Well 2 and 9 contain Fermentas Page Ruler Prestained Protein Ladder. Well 3 and 10 contain the cell lysate. Well 10 contains the cell lysate because the lysate did not enter well 3 cleanly. Well 4 contains the sample after centrifugation. Well 5 and 6 contain the flow through and wash respectively. Well 7 and 8 contain Elution #1 and Elution #2. Figure 7B shows the dried gel. The gel is broken into pieces because the vacuum was opened during the drying process before turning it off which caused a pressure change and broke the gel. Figure 8 shows the Molecular Weight standard of the Fermentas Page Ruler Prestained Protein Ladder.
Discussion:
In the protein expression part of the lab, the results can simply be seen in Figures 1, 2, and 3. Figure 2 shows the cultures are purple, which means these bacteria did take up the purple protein and are expressing it. However, some sources of error did occur prior to this in the bacteria plates. The experimental plate grew bacteria colonies but they were not purple. Since colonies grew on the ampicillin resistant plate that means the DNA was transformed because the bacteria survived. However, a possible reason why the bacteria were not purple could be because the purple gene might not have been expressed at the time but was still part of the bacteria’s DNA. As a result, purple bacteria from another group’s plate was used instead to start the starter culture. The “fun” plate did not grow any bacteria because it was barely coughed on twice. Coughing directly did not place the bacteria on the plate and explains why nothing grew on this plate. The second portion of the lab can be summed up from Figure 4 as it shows Elution#1 and Elution #2. Elution#1 is slightly purple and Elution #2 is clear. The reason for this difference is because Elution #1 is intended to contain most of the purple protein while Elution #2 carries only the protein that failed to wash through in Elution #1.
The gel electrophoresis shows the results of this lab. The gel shows the six samples taken throughout the lab. One source of error is that during the drying process the gel broke. This is because the vacuum was opened while the drying process was still running and caused a pressure change to force the gel to break. In Figure 7, Sample 1 was the most dense as it contained the cell lysate. Well 3 and 10 contain the cell lysate and show the most bands because it contains the most proteins from all of the parts of the cell. One source of error occurred here when placing the cell lysate in Well 3 in which the sample did not properly enter the well because the cell lysate was too thick. Therefore, Well 10 was also used for the cell lysate to try and fix this error. Well 7 shows Elution #1 and a clear distinct band is present, which is the protein of interest. Since only one band exists under Well 7, it can be said that the purity of the final protein is 100%. Well 8 (Elution #2) has no distinct bands because very little protein was present in the elution as intended. Figure 8 shows the Molecular Weight standard of the Fermentas Page Ruler Prestained Protein Ladder. Using this figure it was determined that the approximate molecular weight of the protein in Well 7 (sample 5) is 25 kDa. Using an online convertor, it was found that 25kDa is equal to 4.1535 *10^-12 grams. This was multiplied by Avagadro’s number (6.022*10^23) to find the molecular weight of the protein which was 24999.4 g/mol. This molecular weight is rather close to the scientifically observed molecular weight of 25794.2 g/mol proving that the band in Well 5 is in fact our protein of interest. Thus, despite some sources of error, the gel electrophoresis was still successfully used to analyze the results and determine the molecular weight of the purified protein.
Conclusion:
The lab illustrated how proteins can be isolated and amplified to later be used as potential drug targets. The objective was achieved as a recombinant protein, gbr22, was overexpressed using E. coli, the protein was purified, and a gel electrophoresis was completed to analyze the protein samples. The immediate next step for this lab would be to overexpress and purify other proteins that could be tested as a potential drug targets against various top 15 ligands found in GOLD.
Citations:
1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods2008,5 (2), 135-46
4/18/11
Bacterial Protein Expression, Purification, and Characterization
Objective/Introduction:
Biotechnology has greatly progressed in the last few decades especially in the field of molecular genetics. Technology has been used for protein expression to obtain amplified quantities of proteins that were once previously difficult to acquire. E. coli is often the bacteria of preference as it is harmless to adults. Bacteria are used as expression hosts by transforming the protein of interest in a plasmid vector for the bacteria to incorporate in their own DNA. Bacteria are used for gene expression to amplify the protein of interest[1]. There are several purification strategies to isolate the protein. One such strategy is chromatography in which the solution containing the protein flows through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated to elute the protein from the column. Other protein purification methods include ultracentrifugation and extraction. Expanding technology has clearly led to numerous methods to allow for protein production and purification[2].
In these labs, the purpose to overexpress a recombinant protein (gbr22 purple protein) in bacteria, purify the protein, and use a gel electrophoresis to analyze the protein samples. The importance of these labs is that it builds a foundation to amplify and purify proteins that could be used as potential drug targets in the Virtual Drug Screening research stream.
Materials and Methods:
Several safety precautions were taken during these labs. Gloves, goggles, and lab coats were used throughout because toxic chemicals were used. Also, all biohazardous waste was treated and disposed of properly.
In the first part of this lab, the first step was to transform E. coli bacterial cells with gbr22 protein. 3 plates were used including, one experimental plate with DNA, one Control plate that has no DNA, and one plate for ‘fun’ with bacteria from coughing on the plate. The SOC mixture that either contained the bacteria or did not was accordingly added to the appropriate plate. The starter culture was then grown using LB that was supplemented with 100 mg/mL ampicillin. The starter cultures were transferred to Erlenmeyer flasks and set in the incubator to grow over night. After the cultures were purple, the cells were harvested. Next the cells were resuspended in phosphate buffered saline by adding lysozyme to a final concentration of 1 mg/ml.
In the second portion of the lab, the protein was purified. First the E. coli were lysed with the lysozyme added in the first part of the lab. The lysate was then clarified and labeled as sample 2. The lysate was then syringe filtered to discard large particulate matter. The protein bounded to the resin using Ni-NTA resin/buffer mix. The resin and buffer were then transferred to column chromatography and the resulting sample was labeled as sample 3. The Ni-NTA was then washed through the column with the wash buffer and the resulting sample was labeled as sample 4. The gbr22 protein was released from the Ni-NTA by running the column with the elution buffer. The resulting sample was labeled as Elution 1 and the process was run again to obtain Elution 2. The Nanodrop spectrophotometer was used to estimate the concentration of the final purified protein.
In the last portion of the lab, the protein was characterized. The SDS-page gel samples were prepared and assembled in the electrophoresis module. The wells were loaded using a needle and syringe to clear out the wells and a micropipette to add the samples. Next, the gel was stained by adding imperial protein stain. After a short waiting period, the gels were destained and placed in the orbital shaker overnight. The gel was placed on Whatman filter and Saran Wrap. Lastly, the gel was dried in a vacuum in the Biotech lab.
Results:
Sample 1 (Well 3 and 10): Cell Lysate
Sample 2 (Well 4): After Centrifugation
Sample 3 (Well 5): Flow Through
Sample 4 (Well 6): Wash
Sample 5 (Well 7): Elution 1
Sample 6 (Well 8): Elution2
Protein Ladder (Well 2 and 9)
Figure 1 shows the three plates from part 1 of the lab including the control plate, experimental plate, and fun plate. No bacteria grew on the control plate as intend. Bacteria colonies are present in the experimental plate but they are not seen as purple because they were not expressing the purple protein at the time. The fun plate failed to grow any bacteria on it. Figure 2 shows the two cultures of purple bacteria that grew in the incubator overnight. Figure 3 shows the cell pellet after centrifugation. Figure 3A shows a protein pellet that weighs 0.36 g and Figure 3B shows a protein pellet that weighs 0.24 g. Figure 4 shows Elution 1 and 2 acquired after protein purification. Figure 5 and 6 shows the absorbance spectrum at 280 and 574 nm for the protein. At 280 nm the yield for the average of the trials was 0.662 mg. The yield for the maximal wavelength (574 nm) was 0.201 mg.
Beers Law: A = εlc
Yield for 280 nm Wavelength
A = Average absorbance at 280 nm = (0.22 + 0.225) / 2 = 0.2225
ε = Extinction Coefficient = 39100 M^-1 cm^-1
l = Path Length = 1 cm
Molecular Weight = 25794.2 g/mol
0.2225 = (39100 M6-1 cm^-1) (1 cm) (c)
c = (5.69 * 10^-6 mol/L) (25794.2 g/mol) (1 mg/mL)
c = 0.147 mg/mL
Volume of protein in Elution #1 = 4.5 mL
Yield = (4.5 mL) (0.147 mg/mL)
Yield = 0.662 mg
Yield for 574 nm Wavelength
A = Average absorbance at 574 nm = (0.16 + 0.25) / 2 = 0.205
ε = Extinction Coefficient = 118300 M^-1 cm^-1
b = Path Length = 1 cm
Molecular Weight = 25794.2 g/mol
0.205 = (118300 M6-1 cm^-1) (1 cm) (c)
c = (1.73 * 10^-6 mol/L) (25794.2 g/mol) (1 mg/mL)
c = 0.0447 mg/mL
Volume of protein in Elution #1 = 4.5 mL
Yield = (4.5 mL) (0.0447 mg/mL)
Yield = 0.201 mg
Figure 7A show the gel before it is dried. Well 2 and 9 contain Fermentas Page Ruler Prestained Protein Ladder. Well 3 and 10 contain the cell lysate. Well 10 contains the cell lysate because the lysate did not enter well 3 cleanly. Well 4 contains the sample after centrifugation. Well 5 and 6 contain the flow through and wash respectively. Well 7 and 8 contain Elution #1 and Elution #2. Figure 7B shows the dried gel. The gel is broken into pieces because the vacuum was opened during the drying process before turning it off which caused a pressure change and broke the gel. Figure 8 shows the Molecular Weight standard of the Fermentas Page Ruler Prestained Protein Ladder.
Discussion:
In the protein expression part of the lab, the results can simply be seen in Figures 1, 2, and 3. Figure 2 shows the cultures are purple, which means these bacteria did take up the purple protein and are expressing it. However, some sources of error did occur prior to this in the bacteria plates. The experimental plate grew bacteria colonies but they were not purple. Since colonies grew on the ampicillin resistant plate that means the DNA was transformed because the bacteria survived. However, a possible reason why the bacteria were not purple could be because the purple gene might not have been expressed at the time but was still part of the bacteria’s DNA. As a result, purple bacteria from another group’s plate was used instead to start the starter culture. The “fun” plate did not grow any bacteria because it was barely coughed on twice. Coughing directly did not place the bacteria on the plate and explains why nothing grew on this plate. The second portion of the lab can be summed up from Figure 4 as it shows Elution#1 and Elution #2. Elution#1 is slightly purple and Elution #2 is clear. The reason for this difference is because Elution #1 is intended to contain most of the purple protein while Elution #2 carries only the protein that failed to wash through in Elution #1.
The gel electrophoresis shows the results of this lab. The gel shows the six samples taken throughout the lab. One source of error is that during the drying process the gel broke. This is because the vacuum was opened while the drying process was still running and caused a pressure change to force the gel to break. In Figure 7, Sample 1 was the most dense as it contained the cell lysate. Well 3 and 10 contain the cell lysate and show the most bands because it contains the most proteins from all of the parts of the cell. One source of error occurred here when placing the cell lysate in Well 3 in which the sample did not properly enter the well because the cell lysate was too thick. Therefore, Well 10 was also used for the cell lysate to try and fix this error. Well 7 shows Elution #1 and a clear distinct band is present, which is the protein of interest. Since only one band exists under Well 7, it can be said that the purity of the final protein is 100%. Well 8 (Elution #2) has no distinct bands because very little protein was present in the elution as intended. Figure 8 shows the Molecular Weight standard of the Fermentas Page Ruler Prestained Protein Ladder. Using this figure it was determined that the approximate molecular weight of the protein in Well 7 (sample 5) is 25 kDa. Using an online convertor, it was found that 25kDa is equal to 4.1535 *10^-12 grams. This was multiplied by Avagadro’s number (6.022*10^23) to find the molecular weight of the protein which was 24999.4 g/mol. This molecular weight is rather close to the scientifically observed molecular weight of 25794.2 g/mol proving that the band in Well 5 is in fact our protein of interest. Thus, despite some sources of error, the gel electrophoresis was still successfully used to analyze the results and determine the molecular weight of the purified protein.
Conclusion:
The lab illustrated how proteins can be isolated and amplified to later be used as potential drug targets. The objective was achieved as a recombinant protein, gbr22, was overexpressed using E. coli, the protein was purified, and a gel electrophoresis was completed to analyze the protein samples. The immediate next step for this lab would be to overexpress and purify other proteins that could be tested as a potential drug targets against various top 15 ligands found in GOLD.
Citations:
1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46
2) Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/ (accessed April 17, 2011)